The anticancer activity of Euphorbia lactea Haw. (E. lactea) has been observed by our lab and other research groups; however, the identity of the bioactive compounds harboring the anticancer effect remains unknown. Here, we report the first isolation of four triterpenoidal compounds, i.e., friedelin [1], friedelan-3β-ol [2], taraxerol [3], and friedelan-3α-ol [4], from the n-hexane fraction of the E. lactea extract. The cytotoxic activities of these compounds were investigated in several cancer cell lines, including HN22, HepG2, HCT116, and HeLa. These compounds exhibited a dose-dependent cytotoxic activity against HN22, HepG2, and HCT116, while the marginal cytotoxic effect was observed in HeLa cells. Among the four bioactive compounds, compound 2 exhibited the most prominent anticancer effect against HN22 cells. Flow cytometry analysis of HN22 cells treated with the compound revealed that compound 2 induced a cell cycle arrest at the S-phase, while apoptosis was not induced at the same concentration and exposure time. In summary, our results highlighted E. lactea as an attractive candidate for anticancer research and identified compound 2 as a chemical constituent of E. lactea harboring anticancer activity.